Journal of neuroscience methods · 2025
A comprehensive protocol for simplified mouse DRG fixation, processing and F4/80 immunohistochemistry: Overcoming common challenges.
СтатьяНазвание и аннотация: Перевод в очереди
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Tay Nicolette, Alshammari Ammar, Kaur Simranpreet, Pettit Allison, Mu Erica, Reid Anna, Winkler Ingrid, Vetter Irina, Starobova Hana
Аннотация
BACKGROUND: Dorsal root ganglia (DRGs) contain the cell bodies of sensory neurons and non-neuronal cells that play a role in the pathophysiology of painful inflammatory conditions, such as neuropathic pain. Immunohistochemistry (IHC) is a valuable tool for visualising and quantifying immune cell markers in DRGs, providing important insights into these mechanisms. However, isolating DRGs while preserving cell morphology for IHC staining is technically challenging due to their small size and location within the spinal column.
OBJECTIVE: Using F4/80, a pan monocyte-macrophage marker, we present an optimised protocol for the fixation, harvesting, processing, and IHC staining of formalin-fixed-paraffin-embedded (FFPE) mouse DRGs. This method is designed to maintain tissue integrity and ensure compatibility with downstream histopathological analysis.
NEW METHOD: The entire spinal column of mouse was fixed in 10 % neutral-buffered formalin at room temperature for 24 h before DRG isolation. DRGs were processed for 9 h, and antigen retrieval was performed using proteinase K.
RESULTS: The optimised immersion-fixation approach preserved cellular morphology and antigenicity, ensuring high-quality histological outcomes.
COMPARISON WITH EXISTING METHODS: While transcardial perfusion remains the gold standard for tissue fixation, it is time-intensive, requires training and raises ethical concerns. Our optimised method of whole spinal column fixation with subsequent tissue isolation is non-invasive and reduces the time between death and fixation in comparison to post-isolation fixation. Additionally, it delivers histological quality likely comparable to that of perfusion-based techniques.
CONCLUSION: This protocol is supported by a grading system to help evaluate variables and select conditions best suited to their experimental goals.
OBJECTIVE: Using F4/80, a pan monocyte-macrophage marker, we present an optimised protocol for the fixation, harvesting, processing, and IHC staining of formalin-fixed-paraffin-embedded (FFPE) mouse DRGs. This method is designed to maintain tissue integrity and ensure compatibility with downstream histopathological analysis.
NEW METHOD: The entire spinal column of mouse was fixed in 10 % neutral-buffered formalin at room temperature for 24 h before DRG isolation. DRGs were processed for 9 h, and antigen retrieval was performed using proteinase K.
RESULTS: The optimised immersion-fixation approach preserved cellular morphology and antigenicity, ensuring high-quality histological outcomes.
COMPARISON WITH EXISTING METHODS: While transcardial perfusion remains the gold standard for tissue fixation, it is time-intensive, requires training and raises ethical concerns. Our optimised method of whole spinal column fixation with subsequent tissue isolation is non-invasive and reduces the time between death and fixation in comparison to post-isolation fixation. Additionally, it delivers histological quality likely comparable to that of perfusion-based techniques.
CONCLUSION: This protocol is supported by a grading system to help evaluate variables and select conditions best suited to their experimental goals.